Antibodies specific for intracellular domain of protein tyrosine phosphatase

Abstract

Antibodies to intracellular domains of two or more kinds of protein tyrosine phosphatases, methods for generation thereof and cells producing these antibodies are disclosed. The antibody of the present invention may have specificity to intracellular domains of phosphatase subunits of both of LAR and CD45, and may be useful for analysis and quantitative determination of PTPs, identification and detection of novel PTPs, and for obtaining novel phosphatases by cloning and the like, as well as for developing useful diagnostic methods of insulin resistance and NIDDM, for prophylaxis and diagnosis of various disease states of syndrome X that is based on insulin resistance, and for prophylaxis and diagnosis of onsets of arteriosclerosis and cardiac diseases.

1. A monoclonal antibody having specificity to both an intracellular domain of LAR and an intracellular domain of CD45. 
     
     
       2. The antibody according to  claim 1  having specificity to phosphatase domains of protein tyrosine phosphatases. 
     
     
       3. The antibody according to  claim 1 , which is generated using a polypeptide that is encoded by a nucleotide sequence set forth in SEQ ID NO: 1. 
     
     
       4. The antibody according to  claim 1  wherein the antibody is generated using a GST-LAR phosphatase domain fusion protein as an immunogen. 
     
     
       5. The antibody according to  claim 4  wherein the GST-LAR phosphatase domain fusion protein is produced by: culturing  Escherichia coli  transformed or transfected with an expression vector comprising a coding region of GST gene and a coding region of a phosphatase domain of LAR gene at 20-30° C. for 16-24 hours; and isolating the fusion protein from the culture fluid and/or bacterial cells. 
     
     
       6. The antibody according to  claim 5  wherein the GST-LAR phosphatase domain fission protein is further purified based on an affinity to a support carrying glutathione, and the elution of said fusion protein from the support is performed by boiling in the presence of a detergent. 
     
     
       7. The antibody according to  claim 4  wherein screening of the antibody that was generated using the GST-LAR phosphatase domain fusion protein as an immunogen is performed using said fusion protein. 
     
     
       8. A monoclonal antibody having specificity to an intracellular domain of a protein tyrosine phosphatase, which is produced by a hybridoma with Accession No. FERM BP-6344. 
     
     
       9. The antibody according to  claim 1  having a molecular weight of about 146 kDA. 
     
     
       10. A hybridoma cell line that produces the monoclonal antibody according to  claim 1 . 
     
     
       11. A hybridoma cell line with Accession No. FERM BP-6344. 
     
     
       12. A method for generating an antibody according to  claim 1 , comprising the step of:
 immunizing an animal with a GST-LAR phosphatase domain fusion protein;  
 preparing a hybridoma cell line from an antibody-producing cell obtained from the immunized animal; and  
 producing a monoclonal antibody from the hybridoma cell line.  
 
     
     
       13. The method according to  claim 12  wherein the GST-LAR phosphatase domain fusion protein is produced by: culturing  Escherichia coli  transformed or transfected with an expression vector comprising a coding region of GST gene and a coding region of a phosphatase domain of LAR gene at 20-30° C. for 16-24 hours; and isolating the fusion protein from the culture fluid and/or bacterial cells. 
     
     
       14. The method according to  claim 13  wherein the GST-LAR phosphatase domain fusion protein is further purified based on an affinity to a support carrying glutathione, and the elution of said fusion protein from the support is performed by boiling in the presence of a detergent. 
     
     
       15. The method according to  claim 12 , further comprising the step of:
 screening antibodies generated in the producing step using said GST-LAR phosphatase domain fusion protein.